Neuronal growth factors, neurotrophins and memory deficiency

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Behavioural Brain Research 66 (1995) 129-132

BEHAVIOURAL BRAIN RESEARCH

Neuronal growth factors, neurotrophins and memory deficiency G e m m a Calamandrei a'*, Enrico Alleva b Reparto di Psicologia Comparata and b Reparto di Fisiopatologia Comportamentale, Laboratorio di Fisiopatologia OS, Istituto Superiore di Sanita', Viale Regina Elena 299, 1-00161, Rome, Italy Accepted 15 August 1994

Abstract

CN S and PNS ontogenesis are regulated by various proteic factors, and the best characterized of which still remains Nerve Growth Factor (NGF), a molecule exerting trophic, tropic (i.e. directing growing axons toward NGF-releasing target {issue) and differentiative effects on a number of neural and non-neural (e.g. mast-cells) cell lines. Other Growth Factors (GFs), called 'neurotrophins' (BDNF, NT-3, NT-4, NT-5) also exert similar effects on specific neural cell population. Other GFs (EGF, TGFs, IGFs, FGFs) share these growth-promoting properties with the neurotrophins. NGF appears to regulate specifically the postnatal maturation of the CNS cholinergics in altricial rodents. In adults, cholinergic neurons show retrograde transport for NGF and degeneration of cholinergic neurons after fimbria-fornix transection is prevented by NGF infusion, suggesting a role for NGF in maintaining normal cholinergic function in adulthood. However, peptidergic neurons (e.g. SP-positive cells) seem also to be influenced by perinatal NGF administration, indicating that the spectrum of NGF actions is wider than previously reported. In recent years we investigated the role of NGF in controlling behavioural maturation in the early postnatal period by comparing NGF effects with those of related and non-related neurotrophins (EGF, basic FGF, IGF-1, Transforming GF-alfa). We found that a single intracerebral injection of NGF accelerates cholinergic maturation on postnatal day (PND) 20, as shown by the enhanced reactivity to the muscarinic blocker scopolamine. Scopolamine-induced hyperactivity, normally appearing at the end of the third week, emerges already at PND 5 following NGF administration on PND 2 and 4. Learning and retention capabilities are also influenced by intracerebral NGF in a passive avoidance task (PND 11-12). In the latter, sequestration of endogenous NGF by NGF-antibody administration prevents the 24-h retention normally present on PND 15. KZv words: Nerve growth factor; Neurotrophic factor; Cholinergic system; Neurobehavioural development; Learning and memory; Rodent

There has been a growing acceptance in recent years of the idea that the maturation of the sensory and perceptual capacities of developing mammals should be analysed in greater detail and that those should be related to the ontogenesis of learning and memory in each sensory channel. The characterization of the sequential development of response repertoires has expanded the spectrum of behaviours that can be acquired, indicating that the learning of complex instrumental tasks is possible in the first days of life in spite of the apparent immaturity of the central nervous system at birth. Different memory systems seem to mature at different times in the developing organism in ways which are related to the needs of the organism at a particular time of its life. Whereas learning paradigms designed as to exploit a consistent early behavioural pattern such as suckling are easily acquired and retained at a very * Corresponding author. 0166-4328/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 4 3 2 8 ( 9 4 ) 0 0 1 3 3 - 2

early postnatal age, the ability to perform tasks requiring response suppression and good spatial orienting capacities is not attained until weaning time. In general, some conditioning and learning capabilities seem to emerge before retention capabilities [ 13 ]. As concerns the neural mechanisms that may be responsible for these ontogenetic trends, considerable attention has been devoted to the maturation of cholinergic (particularly muscarinic) regulatory systems. Specifically, there is ample behavioural, pharmacological and biochemical evidence pointing to a rapid maturation of several cholinergic functions around the end of the third postnatal week in altrieial rodents such as rats and mice. At this stage, cholineacetyltransferase (CHAT) activity reaches adult-like levels in the rodent forebraln and animals respond in the adult fashion to cholinergic antagonists, such as scopolamine, with a dramatic increase in locomotor activity [1 ]. Some cholinergic functions, however, appear

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to be functional already at an earlier postnatal stage. For example scopolamine, a muscarinic cholinergic antagonist, produces a paradoxical depressant effect on response latency to choice in a T-maze task in rat pups as young as 7 days, while impairing suckling behaviour in 5-day-old rats and mice [16,17]. Moreover, acquisition of passive avoidance is already present in rats and mice at the end of the first postnatal week or a few days after and is facilitated at this age by physostigmine, a cholinergic stimulant acting via cholinesterase inhibition. This gradual, rather than abrupt, maturation of cholinergically mediated behavioural functions appears to be related to the ontogenesis of septo-hippocampal integration; this is sugggested, for example by Blozovski's correlational study of the development of response suppression and the functional maturation of septohippocampal connections as analysed by intracerebral treatments with muscarinic and nicotinic cholinergic antagonists [4]. Growth and differentiation of nerve cells is profoundly influenced by a number of proteic factors, known as neurotrophic factors. Nerve Growth Factor (NGF) is the best characterized among neurotrophic factors and has been considered for decades a survival and growth-promoting agent for sympathetic neurons and chromaffin cells [ 11 ]. Since the discovery that N G F affects the development of basal forebrain cholinergic neurons, many other neurotrophic factors have been isolated and characterized, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and neurotrophin-3, -4, and -5. All of these molecules are structurally related and seem to act through the same class of receptors in the CNS. NGF, B D N F and NT-3 are able to sustain the survival of distinct, but overlapping, sets of neurons in vitro. These three related neurotrophic factors, now named neurotrophins, may play a parallel as well as a reciprocal role in CNS development, exerting their survival, trophic and tropic action mainly on forebrain cholinergic areas. Also during the past decade, molecules which were previously known for their effects on non-neural tissues have been found to exert neurotrophic effects in cell culture assays. This group of molecules includes the fibroblast growth factor (FGF), the epidermal growth factor (EGF), and the insulin-like growth factors (IGFs) [2]. In the present survey, we will focus on the N G F role in neurobehavioural development of altricial rodents. Indeed an impressive number studies points to N G F as a regulator of cholinergic system development [ 8,9]. Specifically, high N G F levels have been found both in regions innervated by the magnocellular cholinergic neurons of the basal forebrain and in areas containing the cell bodies of these neurons, such as the septum. It has been suggested that the differentiation of magnocellular cholinergic neurons is regulated by N G F retrogradely transported from the hip-

pocampus and the neocortex [10]. The temporal correspondence of increases in ChAT activity and N G F levels during postnatal development strongly supports this hypothesis. Indeed, the activity and the expression of ChAT in the basal forebrain and the caudate-putamen of newborn rats are increased by exogenous N G F [ 12] and decreased by anti-NGF administrations [19]. Cholinergic areas in the CNS of developing rats appear to respond with high regional specificity to exogenous N G F administration. Increasing ChAT activity levels are considered a reliable marker of cholinergic system maturation, and exogenous N G F administration at the brain level somehow mimics endogenous N G F production from target cholinergic areas. As a result, the rapidly changing, regionally specific N G F sensitivity of cholinergic tissues can be easily explained in terms of interference with on-going maturational processes occurring among different brain areas, which use N G F to promote and to guide neuronal growth and/or phenotypic cholinergic expression. Behavioural data point to an anticipation of the emergence of behavioural responses under cholinergic control as a result of early N G F treatment. In mice, a single intracerebroventricular (i.c.v.) administration of N G F around the weaning period has been shown to enhance the hyperactivity response produced by scopolamine, without modifying baseline activity. A single intrahippocampal injection of N G F in rats (PND 8 or 13) accelerated by 5 days the development of spontaneous alternation. Recently, we were able to anticipate the appearance of scopolamine-induced hyperactivity in mice (which normally sets in at the end of the third postnatal week in altricial rodents) to PND 5, by injecting N G F i.c.v, on PNDs 2 and 4. In addition, N G F pretreatment enhanced the scopolamine blocking effect on nipple attachment recorded on pd 5, without altering any other components of the suckling behaviour [5]. In much simpler words, it is possible to shift backwards the first appearance of the second (postweaning) period of cholinergic maturation by simply exposing the developing brain to an unnaturally high amount of exogenous NGF. It has been hypothesized that the NGF-mediated increase in cholinergic neuron metabolism is associated with an accelerated formation of cholinergic synaptic contacts or with an earlier onset of synaptic function. Indeed the behavioural findings are supported by recent in vitro evidence, indicating that exposure to N G F of telencephalic neurons in primary cultures affects the density of muscarinic cholinergic receptors. Exogenous N G F retrogradely transported to the septum appears to stimulate the development of cholinergic innervation over the entire hippocampal formation through direct trophic/differentiative action and/or synaptic remodelling of the cholinergic forebrain populations [7,12]. Quantitative and/or qualita-

G. Calamandrei, E. Alleva / Behavioural Brain Research 66 (1995) 129-132

tive changes in brain muscarinic receptor populations upon early exposure to N G F could explain both the enhancement of some scopolamine effects which normally occur at an early postnatal stage, and the dramatic anticipation of the hyperactivity response to the cholinergic blocking agent. The involvement of CNS cholinergic pathways in learning and memory processes has suggested a role for N G F in cognitive functions. Intraventricular infusion of N G F in aged rats is associated with improved spatial learning and reversal of cholinergic neuronal atrophy [6]. While systemic administration of N G F in the first ten days of postnatal life accelerates sensorimotor maturation of mice [3 ], early postnatal N G F treatment has proven to be unable to modify the rather poor retention performances of neonatal mice in an odor-aversion learning task, though an alteration of pups' olfactory preferences appeared to be induced by N G F exposure. We (submitted paper) explored the passive avoidance (PA) learning profile of 11-12-dayold mice, including effective acquisition without 24h retention, in order to assess the effects of N G F injected intracerebrally on P N D 9. In the task employed, mouse pups had to withold a step-down response from a vibrating platform placed in the center of a metal grid in order to avoid a mild footshock. We found that N G F enhanced unconditioned response suppression, and affected PA performance in the retest session. These results provided evidence for the appearance in N G F pups of 11-12 days of a 24-h PA retention, which was absent in untreated animals. Moreover, previous non-reinforced exposure to the context exerted a marked disruptive effect on the PA performance of N G F pups. It should be mentioned that learning impairment following non-reinforced exposure to the task contingencies (so called latent inhibition) has not been found in rodents before day 17-18, and seems to depend on the integrity of septal and hippocampal structures. Studies still in progress indicate that immunological sequestration of endogenous N G F by means of early antiN G F treatment prevents the 24-h retention of both stepdown and step-through passive avoidance in 15-day mice [ 14]. As a whole the N G F effects here reported are compatible with an accelerated maturation of cholinergic functions following N G F treatment. It cannot be excluded that future research will evidence similar effects on retention performances after early administration of other neurotrophins and/or neuronal growth factors. To date, we found that basic FGF, the administration of which in vivo rescues axotomized cholinergic neurons of the basal forebrain in an action similar to that of NGF, exerts NGF-like accelerating effects on mouse sensorimotor development [ 15]. Moreover, early intracerebral treatment with I G F - I increases ultrasonic vocalization rate in 8-day mice, thus

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suggesting a role for this molecule in the maturation of neural mechanisms involved in the ontogenesis of emotional responses to stressful situations. The increasing body of knowledge about the biological role of neuronal growth factors in mammalian ontogenesis could have profound repercussions on the interpretation of physiological rules of development. Existing data support a model in which a number of related, powerful proteic regulators orchestrate mammalian development, leading sometimes to dramatic changes in the normal ontogenetic pattern.

Acknowledgements This research was supported as part of the Sub-project on Neurobehavioural Pathophysiology (Project of Noninfections Pathology) of the Istituto Superiore di Sanita', and by the National Research Council (CNR) Target Project "Prevention and Control of Disease Factors, subproject Stress". We are grateful to A. Valanzano, L. Ricceri and D. Santucci for their contribution in data collection.

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